The bacterial-mediated diarrheal diseases include cholera, typhoid fever, traveler's diarrhea and acute diarrheal illness in infants. The diseases are basically of two types, invasive and non-invasive gastroenteritis. Typhoid fever is representative of the invasive type which is characterized by invasion of the intestinal mucosa by the pathogen. In the non-invasive type, the symptoms are effected by a bacterial toxin which stimulates an enormous increase in the secretory activity of the cell lining the small intestine causing an acute loss of body fluid.
In the last decade, E. coli has been shown to be the agent responsible for a large portion of previously undiagnosed diarrheal disorders. It has also been determined that E. coli-mediated diarrheal diseases affect not only humans but also agricultural animals, especially cattle and hogs. The newborn of the species is particularly susceptible, and the agricultural industry suffers sizable loss of livestock each year from outbreaks of these diseases.
The E. coli-mediated diarrheal diseases are of the non-invasive type, effected by one or more toxins (or entrotoxins) made by pathogenic strains of E. coli. Two types of toxin, classified as heat-labile (hereinafter LT) and heat-stable (hereinafter ST) have been found in pathogenic E. coli strains causing diarrheal diseases in humans and animals (Sack, R. B., Ann. Rev. Microbiol., 29, 333 (1975)). The genes controlling the production of these toxins are located on plasmids (Gyles, C., et al, J. Infect. Dis., 130, 40 (1974)). In particular, a plasmid designated pCG86 has been isolated and extensively characterized. Plasmid pCG86 is a naturally-occurring recombinant plasmid which contains the genes for LT and ST and several genes for drug resistance. LT is antigenic and partially cross-reactive with anti-cholera antibodies while ST is poorly antigenic. LT is a high molecular weight protein composed of two types of subunits, one (B) having a molecular weight of 11,000 and the other (A) a molecular weight of 25,000 (Dallas, W. S. and Falkow, S., Nature, 277, 406 (1979)). ST has a molecular weight of less than 10,000. Two different types of ST have been discovered, e.g., one ST is active in infant mice, whereas the other is not.
Plasmids have been isolated from pathogenic E. coli carrying drug resistance genes and genes for producing the toxins. (So, M., et al, J. Bacteriol., 128, 463 (1976) and Gyles, C. L., et al, Science, 198, 198 (1977). Such plasmids are naturally-occurring recombinant plasmids. The gene coding for LT has been cloned in a multi-copy plasmid (So, M., et al, Infec. Immun., 21, 405 (1978)). Variants of pCG86 have been produced bearing mutations in the LT gene or the ST gener. (Silva, M. L. M., et al, Proc. Nat. Acad. Sci. USA, 75, 1384 (1978)). Loss of LT toxicity can result from mutation in either the A or B subunit. The presence of a plasmid containing the genes for LT, ST or both has been shown to be insufficient for causing diarrheal disease. A colonization factor, a specialized adherence pili, which is antigenic, is also required. This colonization factor is required for the pathogenic E. coli to adhere and proliferate in the small intestine. The colonization factor gene may be carried on the same or a separate plasmid. For a review of this material, see Broda, P., Plasmids, W. H. Freeman & Co., San Francisco, Ca., pp. 120-122 (1979).
Although the toxins exert their effect in the lumen of the gut and do not enter the bloodstream, it is possible to provide immunity of the IgA type against antigenic proteins produced by resident bacterial strains. Thus, for example, recovered cholera patients are immune to further challenges, and they have been shown to have protective antibodies in their gastrointestinal tracts. Immunity can be generated by promoting antibody formation against the enterotoxins or against the colonization pili. For a review, see Levine, M. M., et al, Microbiol. Rev. 47, 510 (1983). Immunity can be provided by administering a live vaccine in the form of a mutant bacterial strain producing an altered toxin, lacking toxic effects but able to elicit antibodies capable of cross reacting with and inactivating the wild-type toxin. The foregoing approach has been applied successfully in the case of cholera, which produces an antigenic toxin (Honda, T. and Finkelstein, R. A., Proc. Nat. Acad. Sci. USA, 76, 2052 (1979)). Prior to the present invention, it was not possible to apply this technique to achieve immune protection against pathogenic E. coli because of the toxicity of both LT and ST and the requirement of an additional colonization factor. The present invention provides a strain of E. coli capable of colonization which produces an immunogenic LT lacking toxicity and a non-toxic ST. As used herein, a gene (or strain) producing a non-toxic ST refers to a gene (or strain containing such gene) which either produces a mutant ST that is non-toxic or does not produce any ST at all. The immunogenic LT is capable of eliciting a protective IgA response when produced by the strain of E. coli which is now nonpathogenic. The present invention also provides a recombinant plasmid, containing genes coding for an immunogenic, non-toxic LT capable of eliciting an antibody which neutralizes naturally occuring toxins.